Loss of heterozygosity (LOH) at chromosomal band 9p21 is one of the few consistent genetic aberrations found in conventional chondrosarcoma. This locus harbours two cell-cycle regulators, CDKN2A/p16/INK4A and INK4A-p14ARF, which are inactivated in various human malignancies. It was therefore hypothesized that this locus also plays a role in the development of chondrosarcoma and this locus was investigated at protein, genetic, and epigenetic levels. Loss of p16 protein expression was detected by immunohistochemistry in 12 of 73 central chondrosarcomas and it correlated with increasing histological grade (p = 0.001). Loss of p16 protein expression was not found in 51 enchondromas, which are presumed to be potential precursors of conventional central chondrosarcoma. LOH at 9p21 was found in 15 of 39 chondrosarcomas (38%) but it did not correlate with loss of p16 protein expression. SSCP analysis of p16 did not reveal any mutations in 47 cases. Also, p14 was not the target of LOH, since it gave no aberrant bands on SSCP. To investigate whether an epigenetic mechanism was operating, methylation-specific PCR was used to look at p16 promotor methylation, which was identified in 5 of 30 tumours. However, this did not correlate with protein expression, or with LOH at 9p21. Cytogenetic data were available in a subset of cases. All tumours that showed chromosome 9 alterations also showed LOH and loss of INK4A/p16 protein expression. It is concluded that although some alterations were found at the DNA level and at the promoter expression level, the lack of correlation between LOH, promotor methylation, and protein expression indicates that a locus other than CDKN2A/p16 must be the target of LOH at 9p21. The correlation between INK4A/p16 protein expression and tumour grade, and the retention of expression in enchondromas, indicates that loss of INK4A/p16 protein expression may be an important event during tumour progression from enchondroma to conventional central chondrosarcoma, and in the progression in grade after recurrence of chondrosarcoma.